This project will study the developmental control of expression of the two types of 5sRNA genes (5sDNA) in the frog Xenopus borealis. Somatic cells produce a single kind of 5sRNA, while oocytes synthesize an additional 5s species. The developmental control mechanism appears to shut off oocytes 5sDNA in somatic cells. Both types of genes have already been purified and cloned, and the complete nucleotide sequences of one somatic 5sDNA clone (Xbsl) and one oocyte 5sDNA clone (Xbo1) are known. A second Xbo clone will be sequenced to investigate in detail the sequence heterogeneity of Xbo repeats suggested by previous work. To determine if there is transcriptional level control of 5sDNA expression, chromatin will be fractionated into transcriptionally active and inactive fractions. Radioactively labelled probes from the sequenced 5sDNA's will be used to determine whether a given gene type is only in the active fraction at developmental times when it is expressed. Differences in the 5sDNA chromatin structure in each fraction will be studied with appropriate enzyme probes. The labelled 5sDNA probes will also be used in attempts to identify specific DNA-binding proteins. Proteins from both oocytes and somatic cells will be fractionated. Each fraction will be assayed for DNA-binding activity by its ability to specifically retain labelled Xbo, or Xbs on filters in the presence of excess amounts of nonspecific DNA. By labelling each type of DNA with a different isotope, a single assay can determine if any protein fraction binds one type of DNA in preference to the other. Since the DNA probes are fully sequenced, any binding sites can be easily localized by sequencing those DNA regions made resistant to nucleases after DNA-protein interaction. Finally, attempts will be made to determine if the DNA-binding proteins play a role in the specific transcription of the 5sDNA's by Xenopus RNA polymerase III.